This competing renewal application builds on our studies in the Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) selected lines and the DBA/2 inbred strain, which suggest that genetic differences in ethanol withdrawal severity are due in part to alterations in the ?-aminobutyric acid (GABA)- modulatory effects of neurosteroids such as allopregnanolone (ALLO), the most potent positive modulator of GABAA receptors. In particular, during ethanol withdrawal, endogenous ALLO levels were reduced, and the anticonvulsant effect of systemically administered ALLO was decreased in WSP and DBA/2 mice, but not in WSR mice. Further, in ethanol withdrawn WSP mice, a reduced sensitivity to ALLO's anticonvulsant effect was also observed when ALLO was infused into specific brain regions within the withdrawal seizure circuit, including the hippocampus. Preliminary electrophysiological experiments in hippocampal slices from ethanol withdrawn WSP mice documented a reduced effect of GABAergic steroids and selective GABAA receptor ligands on GABAA receptor mediated synaptic and tonic currents. Additionally, epileptiform activity in hippocampal slices from ethanol withdrawn WSP mice was reduced by GABAA receptor enhancing ligands. Finally, preliminary experiments with gas chromatography-mass spectrometry (GC-MS) simultaneously quantified hippocampal neurosteroid levels and determined that ethanol injection produced divergent changes in steroids with positive (agonist) and negative (antagonist) effects on GABAA receptor function. Based on these data, the current proposal will adopt a multidisciplinary approach to test the overall hypothesis that a reduction in hippocampal GABAergic function, due to decreased GABAA receptor sensitivity to neurosteroids and a concomitant imbalance in ALLO and related neurosteroids, represents a neurochemical substrate for enhanced susceptibility to ethanol withdrawal severity. Studies will be conducted in WSP and DBA/2 mice, two animal models that are widely used to study genetic liability to ethanol withdrawal in mammals. Aim 1 will use GC-MS to quantify the effect of ethanol withdrawal on multiple endogenous steroid compounds, with agonist and antagonist effects on GABAA receptor function, in the hippocampus at select time points of withdrawal. Aim 2 will use voltage-clamp recording in subregions of hippocampal slices to characterize ethanol withdrawal-induced changes in the pharmacology of synaptic and extrasynaptic GABAA receptors, including alterations in sensitivity to neurosteroids with agonist and antagonist effect and to subunit selective ligands. Aim 3 will integrate information from Aims 1 and 2 and determine if counteracting ethanol withdrawal-induced changes in GABAA receptor properties and in neurosteroid levels rescues the high withdrawal phenotype, measured by epileptiform activity in hippocampal slices and convulsions in vivo. Thus, the proposed experiments will establish the importance of altered hippocampal neurosteroid composition and GABAA receptor pharmacology in mediating ethanol withdrawal severity.

Public Health Relevance

Withdrawal is one important dimension of genetic risk for alcohol dependence and the development of alcoholism, and it is a serious medical consequence in up to 71% of the more than 1.5 million people in the US that are hospitalized each year for dependence-related medical consequences. Significance of the proposed work lies in the use of two genetic animal models of high alcohol withdrawal with similar temporal pattern of withdrawal-related tremor and convulsions to humans, and a multidisciplinary strategy to examine the mechanisms mediating susceptibility to high convulsive behavior during alcohol withdrawal. This information will aid in our understanding of mechanisms fundamental to alcohol withdrawal, and it will help in the development of new strategies for the treatment of alcohol dependence and withdrawal.